JP3393379B2 - Amino acid adsorbent and method for recovering amino acid - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an amino acid adsorbent and a method for recovering amino acids.

[0002]

2. Description of the Related Art Methods for obtaining amino acids include (1) extraction and separation from amino acid mixtures, (2) chemical synthesis, (3)
There is a fermentation method. The extraction and separation method of (1) above is a separation in which an acidic ion exchange resin and a basic ion exchange resin are appropriately combined with a plurality of amino acid mixtures obtained by hydrolyzing an inexpensive protein such as soybean gluten with hydrochloric acid. Repeat the operation to sort out specific amino acids,
Alternatively, it is a method of fractional crystallization by utilizing the difference in the solubility of amino acids. This separation method is not performed at present because it is laborious and it is quite difficult to make a pure product free from other amino acids.

According to the synthesis method of (2) above, the L-form and the D-form must be separated by the optical resolution method in order to form a racemic body. The problem of purity remains. Therefore, in this synthetic method, only limited amino acids such as glycine and methionine are produced at present.

The above-mentioned fermentation method (3) is the current mainstream method, in which a microorganism is cultivated in a medium to accumulate amino acids outside the cells (in the culture broth) and then separated and purified from the culture broth. This separation and purification has an advantage that it can be relatively easily carried out by separation with an ion exchange resin or concentrated crystallization. In the fermentation method, it is important to use a material capable of selectively adsorbing a specific amino acid when extracting the amino acid from the culture solution at the final stage, and various materials have been proposed as such a material. ing. The annual production value of amino acids in the world by fermentation is over 100 billion yen. The production volume is L-
Sodium glutamate (190,000 tons per year), methionine (40,000 tons per year), lysine (30,000 tons per year) are listed in that order.
Lysine is in demand for nutrition next to methionine,
It is commercialized on a large scale next to glutamic acid. Lysine has become more important as a protein resource for feed, and is an amino acid that is expected to develop most in the future as an essential amino acid. However, in spite of the fact that it can be produced in large quantities, the fermentation process is complicated, so development of simple separation and isolation techniques is desired.

As described above, the method for extraction and separation from the amino acid mixture is not practiced at present. However, if an economical and effective method for extracting a specific amino acid is found, this extraction / separation method is also a fermentation method as a method for effectively utilizing or reusing a huge natural protein resource. It is expected that it will become a meaningful method with. That is, if a material that can selectively and efficiently adsorb a specific amino acid from an amino acid mixture is developed, such a material can be used for separating and recovering the specific amino acid, so that it can be used in food manufacturing processes, chemical processes, and It can be expected to be used as an amino acid adsorbent in various industrial processes.

[0006]

As described above, it is desired to develop a technique for separating and recovering a specific amino acid from an amino acid mixture by an efficient and economical method. In order to separate and recover a specific amino acid without adding a chemical reaction to an amino acid mixture, it is necessary to search for a material that has a different affinity for various amino acids and develop a material that can adsorb only the specific amino acid. Is the way. Therefore, an amino acid adsorbent capable of adsorbing a specific amino acid from an amino acid mixed aqueous solution containing various amino acids (hereinafter also referred to as an amino acid-containing aqueous solution) has been developed based on the principle of chromatography. No material has yet been found.

Further, the amino acid adsorbent is packed in a column, and an amino acid-containing aqueous solution is made to flow through the column, or the amino acid adsorbent is brought into contact with the amino acid-containing aqueous solution to adsorb a specific amino acid, and then the adsorbent is adsorbed. If a method can be found that can recover the specified amino acid by an appropriate method, it is expected to be an economical and efficient technique for producing, separating, and recovering the amino acid. No method has yet been found. An object of the present invention is to provide an adsorbent for adsorbing a specific amino acid from an amino acid-containing aqueous solution, and a method for recovering a specific amino acid using the adsorbent.

[0008]

SUMMARY OF THE INVENTION The present inventors have proposed a number of specific amino acids that are adsorbed to these materials when a natural protein or a synthetic polyamino acid is packed in a column and an amino acid-containing aqueous solution is passed through the column. He has been diligently pursuing basic research. As a result, cocoon threads made by silkworms, natural proteins consisting of silk proteins such as silk threads or animal proteins such as wool keratin, or synthetic polyamino acids consisting of homopolymers or copolymers obtained by polycondensation of amino acids, have specific amino acids. The inventors have found that they are efficiently and specifically adsorbed, and have completed the present invention. Further, a silk protein in a variety of forms such as powder, beads, a film, a fiber, or a natural protein such as animal keratin, or a polymeric material composed of a synthetic polyamino acid is packed in a column, and an amino acid-containing aqueous solution is poured into the column. ,
When these materials are brought into contact with an amino acid-containing aqueous solution,
Since specific amino acids are specifically and efficiently adsorbed to these materials, it was also found that coating the surface of the carrier with these materials provides the same adsorption effect as that of the materials themselves.

The amino acid adsorbent of the present invention comprises silk protein or carrier fine particles whose surface is coated with the silk protein , and is present in an aqueous solution containing a plurality of amino acids.
Mainly composed of lysine, arginine and histidine
Basic amino acids, mainly aspartic acid and
It selectively adsorbs acidic amino acids composed of lutamic acid . This silk protein is in the form of powder, beads, film, fiber, or liquid. The silk protein is a concentrated aqueous solution of a water-soluble resin (concentration: for example, sodium carboxymethyl cellulose (hereinafter, C
(Abbreviated as MC), mix with 10-30% by weight),
It may be a powder or micronized fiber prepared by drying to a solid state (which may be freeze-solidified to a solid state if desired) and then pulverizing a solidified resin containing the protein. The silk protein may be in the form of powder, beads,
It may be in the form of a film or fiber.

The silk protein is a silk fibroin fiber.
Silk fibers prepared by immersing and heating and dissolving
Silk fibs obtained by dialysis of aqueous broin solution against pure water
Acid is added to the aqueous solution of loin to adjust the pH to 3 to 3.5 to cause gelation.
Lyophilized and powdered silk protein,
May be a liquid silk protein within the silk gland .

The method for recovering a specific amino acid of the present invention is
An aqueous solution containing multiple amino acids is added to the non-acid adsorbent.
Mainly present in the amino acid-containing aqueous solution by contacting
Basic acid consisting of lysine, arginine and histidine
Mino acids and mainly aspartic acid and glutamic acid
Selectively adsorb the acidic amino acid consisting of
Then, after washing the adsorbent with water, use an acidic aqueous solution
Consists mainly of lysine, arginine and histidine
Basic amino acid is desorbed from the adsorbent and recovered.
Also, aspartic acid was mainly prepared using an alkaline aqueous solution.
And the acidic amino acid consisting of glutamic acid
It is characterized in that it is desorbed and collected.

Here, the acidity of the acidic aqueous solution is preferably pH≈1.0 to 4.0, more preferably pH≈1.0 to 3.5,
The alkalinity of an alkaline aqueous solution is preferably pH≈7.
It is 5 to 13.0, and more preferably pH≈8.0 to 11.0. In the case of acidic, if the pH is less than 1.0 and exceeds 4.0, it is difficult to achieve the intended purpose of desorbing the basic amino acid without damaging the adsorbent. If it is less than 7.5 and more than 13.0, it is difficult to achieve the intended purpose of desorbing the acidic amino acid without damaging the adsorbent.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The amino acid adsorbent of the present invention is described below.
Animal keratin and synthesis, along with constituent silk proteins
Polyamino acids are also described. Silk data <br/> protein in the present invention are, for example, silkworm, silk protein fibers from Kuwako, Antheraea yamamai, acetic, Samia cynthia ricini, silk protein fibers from wild silkworm such Samia Cynthia, silk textiles or fiber assembly, Ru is included. Also, the animal keratin,
For example, keratin fibers of animal fibers typified by wool, keratin fiber products, or fiber aggregates thereof may be included, and further spider thread, collagen, gelatin may also be included.

Instead of the silk protein fiber made by silkworm, liquid silk protein in the silk gland obtained by dissecting the silkworm and taking it out from the body can be used. Using a fibrous sample spun by silkworms is more efficient than using a liquid silk protein sample in the silkworm body in that a large amount of sample can be obtained at any time throughout the year. Further, instead of the above Kiten <br/> natural protein, can be used amino acids as well synthetic polyamino acids by polycondensation. Further, a material obtained by coating the surface of the above-mentioned protein or synthetic polyamino acid on the surface of fine particles made of an organic substance or an inorganic substance can also be used as an amino acid adsorbent.

Natural proteins are composed by polycondensation of various amino acids. The molecular side chain has both a positively charged group such as L-lysine and L-arginine, and a negatively charged group such as L-aspartic acid and L-glutamic acid, and is therefore called an ampholyte. Further, the case <br/> formed polyamino acid, as described below, glycine, alanine, valine, phenylalanine, glutamate, aspartate, leucine, or N- carboxy anhydrides of amino acids such as isoleucine (NCA),
Or amino acid NC of glutamic acid ester or aspartic acid ester whose molecular side chain is blocked with benzyl group etc.
It can be produced by polymerizing A in an organic solvent. These synthetic polyamino acids are non-electrolytes because even if they become polymers, the molecular side chains have no charge. In the above-mentioned synthetic polyamino acid, since all amino acids other than glycine are L-forms in the amino acid raw material, each amino acid may not be represented as L-form.

The silk protein will be described by taking silk fibroin fiber as an example. Silk fibroin fiber, after solidifying this fiber with a water-soluble resin such as CMC, freeze-drying treatment if desired, crushing the solid fiber,
Alternatively, this crushing step can be repeated to use in the form of powder or finely divided fibers. The water-soluble resin is not particularly limited as long as it can be removed by washing the obtained powder with water. It can also be used as a powdery sample prepared from an aqueous solution of silk fibroin fiber. That is, the silk fibroin fiber is immersed in a concentrated aqueous solution of a neutral salt such as lithium bromide or calcium chloride and heated to dissolve, and the resulting silk fibroin aqueous solution is put into a cellulose dialysis membrane and purified by dialysis with pure water. ,
Concentrate by air drying to obtain a concentrated silk fibroin aqueous solution.
Next, acid is added to this silk fibroin aqueous solution to adjust the pH =
After gelling to 3 to 3.5 and freeze-drying at about -20 ° C, it is ground appropriately in a mortar to obtain powdered silk fibroin. This powdered silk fibroin is insoluble in water and can be used as an amino acid adsorbent.

Next, animal keratin will be described by taking wool keratin fiber as an example. Wool keratin can be used as a keratin aqueous solution or an S-carboxymethyl keratin aqueous solution as described below.

First, in order to dissolve wool keratin fibers, intermolecular cystine bonds are cleaved in nitrogen using a reducing agent such as mercaptoethanol or thioglycolic acid to reduce keratin molecules to make them water-soluble. When using mercaptoethanol, it is advisable to carry out the reduction treatment in a urea solution. The concentration of urea is generally 7.5-8.8 mol / L,
It is preferably 7.8 to 8.0 mol / L. When using thioglycolic acid, it is advisable to add 1-4% NaCl. As a reducing agent, for example, when using mercaptoethanol, wool keratin fibers are immersed in an aqueous urea solution having the above concentration, and after degassing, under a nitrogen atmosphere, 45 ° C. or less, preferably
Add mercaptoethanol to wool keratin fibers at a temperature of 20 to 25 ° C (for example, add 3.5 mL of mercaptoethanol to 10 g of wool keratin fibers), and further add about 3
Stir for hours. In this way, the keratin molecule is reduced, and wool keratin having an SH group is obtained. Then, it is dialyzed with pure water to remove urea and excess mercaptoethanol to obtain an aqueous wool keratin solution. The keratin solution can be used as a water-soluble native protein. For example, the aqueous keratin is concentrated by air-drying, and the wool keratin that recombines during the concentration can be collected by filtration, dried, and utilized as a powdered wool keratin,
The remaining aqueous solution concentrated by air drying can be freeze-dried and used as powdery wool keratin as in the case of silk protein fiber.

Further, the wool keratin having an SH group obtained as described above is further reacted with an alkylating agent, for example, a known alkylating agent such as (substituted) alkyl halide to give S- (substituted) alkyl keratin. if it is possible to take advantage that tossed the aqueous solution. This alkylation may be performed according to a known method. As an example, the case where iodoacetic acid is used as the alkylating agent will be described. Iodine acetic acid (for example, 10 to 17 g of iodoacetic acid to 10 g of wool fiber) is added to and reacted with the above-mentioned reduced keratin in nitrogen at a temperature of 20 to 25 ° C. with stirring. After 1-2 hours, the pH is adjusted to about 8.5 and dialyzed with pure water to remove excess iodoacetic acid to obtain an S-carboxymethyl keratin aqueous solution. The S- carboxymethyl keratin solution can be used as water-soluble natural proteins. For example, S-carboxymethyl keratin aqueous solution is spread on the surface of a support such as an organic polymer material such as polyester, polyamide, polypropylene, polyethylene, polystyrene or an inorganic material such as glass, water is evaporated, and the mixture is dried and solidified. Further, if desired, an epoxy compound is allowed to act to make it insoluble in water, whereby a keratin film can be prepared. The support is not particularly limited as long as the film can be easily peeled off after drying. Further, this S-carboxymethyl keratin aqueous solution can be used in the form of powder in the same manner as above.

As described above, the powdery sample obtained from silk fibroin fiber, which is a silk protein, is prepared by dissolving silk fibroin fiber in an aqueous solution of neutral salt and freezing low molecular weight molecules other than silk fibroin with a dialysis membrane. Although dried can be prepared, since the powdered silk fibroin is water soluble, and since it is also soluble if powdered wool keratin obtained as described above, when used as an amino acid adsorbent , Need to be water insoluble.

To render silk fibroin, silk proteins such as silk sericin, or wool keratin water-insoluble,
The solubility in water may be controlled by chemically crosslinking the molecules. In order to make powdery or membranous silk fibroin insoluble in water, it is subjected to a dipping treatment using, for example, 30 to 70% by weight of alcohol such as ethanol or methanol. This treatment makes the aggregate structure between the sample molecules dense, that is, the aggregate density is improved, the crystallinity is increased, and the sample becomes water-insoluble. In order to render silk protein or wool keratin insoluble in water, it may be treated with an intermolecular crosslinking agent such as an epoxy compound or an aldehyde compound. As the epoxy compound, a bifunctional epoxy compound such as ethylene glycol diglycidyl ether, and other trifunctional and polyfunctional epoxy compounds can be used, and as the aldehyde compound, glutaraldehyde or acetaldehyde is usually used. it can.

In addition to the above-mentioned powdery silk fibroin and powdery wool keratin, a natural protein in the form of beads, a film or a fiber which can be used as an amino acid adsorbent, and a synthetic polyamino acid described in detail below are also available. In order to adsorb an amino acid by bringing it into contact with an amino acid-containing aqueous solution, it must be water-insoluble. Also in this case, similarly to the case of powdered silk fibroin, it can be easily made water-insoluble by causing an epoxy compound or an aldehyde compound to act.

[0023] Synthetic polyamino acids which can be used as amino acid adsorbent amino acid is a polymer compound of the polycondensed structures, have imino group to an amino group and a main chain terminus. The synthetic polyamino acids include homopolymers of a single amino acid residue, and copolymers of multiple amino acid residues and the sequence of the amino acid residues is random. The polyamino acid can be synthesized by polymerizing the corresponding amino acid NCA,
It can also be produced by dehydration condensation of amino acids by other methods. A relatively simple method for synthesizing polyamino acids
Te, describing the center the following under amino acid NCA method. For example, NCA of each amino acid was synthesized from amino acids such as glycine, L-alanine, L-valine, and L-phenylalanine, and then polymerized in an organic solvent to obtain a homopolymer (for example, polyglycine, polyalanine, polyvaline). Etc.). The copolymer is obtained by mixing two or more kinds of amino acids NCA and polymerizing them.

[0024] Synthetic polyamino acids, N- carboxy anhydride method (NCA method) (Leuchs, Chem. Ber. 38,852 (1906)), dehydration condensation of amino acids to each other, Ru can be produced by such as a solid phase method of Merrifield. It was synthesized polyamino acid by amino acid NCA method. First, the amino acid NCA is synthesized from the amino acid and the phosgene derivative. After the amino acid NCA crystal is purified, it is dissolved in a solvent such as acetonitrile or dioxane, and a base such as amine or sodium methoxide is added as a polymerization initiator. By proceeding the reaction in an environment where water is not mixed, a synthetic polyamino acid having an average degree of polymerization of several hundreds can be easily obtained. It can also be obtained by immersing a crystal of amino acid NCA in a non-solvent such as hexane or decane, adding a trace amount of water or a base, and polymerizing it in the solid phase (solid phase polymerization). Compared with other synthetic methods, the NCA method can produce large amounts of high-molecular-weight synthetic polyamino acids in a short time.

Among the above amino acids, those having a hydroxyl group in the side chain such as L-glutamic acid and L-aspartic acid are blocked with a benzyl group, a methyl group, an ethyl group or the like when the NCA is produced. There is a need to. By this, if benzylation is performed, L-glutamic acid
It becomes a γ-benzyl ester, and the synthetic polyamino acid made from its NCA becomes poly (γ-benzyl-L-glutamate) (abbreviation: PBLG). Amino acids having a functional group, such as the above-mentioned hydroxyl group, which hinders the reaction when forming NCA, are blocked with a protecting group and then made into a polyamino acid. As the amino acid protecting group, conventionally known ones, for example, carbobenzoxy group, phthalyl group, formyl group,
A benzyl group, a trialkylsilyl group, an acetoacetyl group, a benzenesulfonyl group or the like can be used.
The most preferred of these is a benzyl group from the viewpoint of handling and the fact that the amino acid protecting group can be easily removed if necessary.

An example of synthesizing an amino acid NCA from an amino acid and producing a synthetic polyamino acid based on the amino acid NCA will be described. First, in order to synthesize the amino acid NCA, the amino acid is pulverized in a mortar and placed in a liquid dispersion medium in which the amino acid is dispersed. As the dispersion medium, tetrahydrofuran (THF), ethyl acetate, toluene, dioxane and the like can be used, but a preferable one is excellent in dissolving power of the amino acid NCA to be produced,
It is THF that can be easily distilled off under reduced pressure. Amino acid NCA is synthesized by adding triphosgene, trichloromethyl chloroformate, phosgene gas, thionyl chloride, etc. to a dispersion liquid in which amino acids are dispersed. Among these, triphosgene and trichloromethyl chloroformate are preferably used from the viewpoint of easy handling and convenience in use. The generated amino acid NCA can be obtained, for example, as a THF solution. In this case, THF is distilled off by vacuum distillation to obtain solid amino acid NCA, which is purified by recrystallization from a mixture of ethyl acetate and hexane.

Next, the obtained amino acid NCA is polymerized. As a solvent therefor, acetonitrile, ethyl acetate, toluene, dioxane, etc. can be used, and as a polymerization initiator, butylamine, hexylamine, se
Bases such as c- or tert-butylamine, triethylamine, sodium methoxide can be utilized. Water may be used as the polymerization initiator. Butylamine is one that can be precisely controlled in the degree of polymerization and is easy to handle. A polyamino acid is produced by adding a polymerization initiator to an amino acid NCA solution and allowing it to stand at a temperature of 20 to 50 ° C. for several hours to several weeks. Pour the reaction solution into about 10 times the volume of hexane,
The resulting polyamino acid precipitates upon stirring. A powdery polyamino acid is obtained by filtering the precipitate and drying it.

The characteristics of the synthetic polyamino acid adsorbent thus obtained depend on the kind of amino acid and the degree of polymerization of the synthetic polyamino acid. The degree of polymerization is closely related to the molar ratio of the polymerization initiator and the amino acid NCA monomer, that is, the molar ratio = [the number of amino acid NCA] / [the number of polymerization initiator], and the ideal polymerization If so, this molar ratio corresponds to the average degree of polymerization. In this case, since it is necessary to raise the molecular weight of the synthetic polyamino acid to a certain level or higher, the molar ratios were all set to 200 when synthesizing any of the synthetic polyamino acids, but it is not necessary to limit to this value. However, 100 or more is preferable.

The synthetic amino acid copolymer can be synthesized as follows. Two or more kinds of amino acids NCA selected from amino acids such as glycine, alanine, valine and phenylalanine are dissolved in an organic solvent such as acetonitrile. For example, when using glycine NCA and alanine NCA, glycine NCA and alanine NCA are dissolved in acetonitrile at a molar ratio of 1: 2. Next, butylamine as a polymerization initiator is added in a total amount of [the total number of moles of glycine NCA and the number of moles of alanine NCA] / 200 to carry out the polymerization. In theory, the average degree of polymerization = 200 A copolypeptide is obtained.
The copolypeptides obtained from the above amino acids NCA are produced as water-insoluble copolymers by precipitation in an organic solvent such as acetonitrile.

[0030] A As the amino acid adsorbent, other available natural protein or a synthetic polyamino acid of various forms as described above, the silica-based carrier or other fine particles composed of carrier of the native protein and synthetic poly such as silica gel Those coated with amino acids can be used as well. As other carriers, for example, diatomaceous earth, pumice stone, activated carbon, inorganic carrier fine particles such as activated alumina, carrier fine particles made of natural materials such as cellulose, or polystyrene,
Even fine particles of synthetic organic polymer such as polymethylmethacrylate can be used in the same manner. The shape of the carrier is a spherical particle shape, and finer particles have higher utility value as an industrial material. For example, a commercially available granular column packing material such as silica gel, the surface of which is coated with a polymer material composed of the above-mentioned natural protein or synthetic polyamino acid can be used as an adsorbent. That is, the granular column packing material was immersed in an aqueous solution of the polymer material, allowed to stand for a predetermined time, and then the column packing material was taken out and
After drying at room temperature of -25 ° C, it is dipped in alcohol such as methanol to obtain an adsorbent in which the surface of the column packing material is coated with a polymer material. An adsorbent whose surface is coated with a polymer film having a desired thickness can be prepared by appropriately changing the concentration of the polymer material aqueous solution or by repeating this dipping operation.

In the case of the column packing material coated with the natural protein in this manner, the material coated on the surface (eg, silk protein, wool keratin) may be dissolved in water. Water-insolubility of such silk proteins and wool keratin can be easily carried out by the above-mentioned dipping treatment using alcohol or treatment using epoxy compound or aldehyde compound.

A method for producing a column packing material (silica gel particles) coated with a synthetic polyamino acid will be described below. For example, silica gel particles are added to an aqueous solution obtained by dissolving 0.5 to 2.0% by weight of a synthetic polyamino acid in a mixture of dichloroacetic acid or trifluoroacetic acid and chloroform (chloroform content ratio: 30 to 80% by volume), and the silica gel particles are applied to the particle surface. A synthetic polyamino acid is adhered, and the silica gel particles are collected by filtration with a glass filter, then put into methanol or ethanol and stirred. After filtration, drying under reduced pressure makes it possible to prepare silica gel particles whose surface is coated with a synthetic polyamino acid. Alternatively, a synthetic polyamino acid such as poly (γ-benzyl-L-glutamate) or poly (β-benzyl-L-glutamate) is dissolved in a suitable organic solvent (for example, chloroform or dimethylformamide), and the solution is dissolved in this solution. Put a carrier such as silica gel particles in
Stir gently for about 10 minutes. Next, it is obtained by filtering out with a glass filter and drying for about 1 hour in a drier at 60 to 80 ° C. Synthetic polyamino acids may be soluble only in strong acids such as trifluoroacetic acid and dichloroacetic acid, depending on the types of amino acid residues. In such cases, after dissolving the synthetic polyamino acid in these strong acids,
Chloroform, dimethylformamide, etc. may be added and diluted to such an extent that the dissolved polymer does not precipitate, and this may be used as a synthetic polyamino acid solution for the coating step. When the synthetic polyamino acid on the surface of the particles thus obtained is water-soluble, it is rendered water-insoluble by the water insolubilization treatment described above.

The mosquitoes were once dissolved protein fibers or wool other animal protein fibers obtained from equalizing obtained from an aqueous solution which was prepared by a powder-like sample, the fine fibers obtained by cutting these protein fibers or powder synthesis, By using polyamino acid or the like as an amino acid adsorbent, a specific amino acid can be adsorbed from an amino acid-containing aqueous solution. When a material consisting of natural protein or synthetic polyamino acid is packed in a column and an amino acid-containing aqueous solution is run through the column, the adsorbent obtained from silk protein fiber or powder mainly contains basic amino acids such as lysine, arginine and histidine. It is selectively adsorbed, and with other adsorbents, mainly acidic amino acids such as aspartic acid and glutamic acid are selectively adsorbed.

The amino acid-containing aqueous solution means a plurality of amino acids (for example, Asn, Asp, Arg, Ala, Ile, Gly, Gln, Glu,
Cys, Ser, Tyr, Trp, Thr, Val, His, Phe, Pro, Met,
Lys, Leu, etc.). Since the aqueous solution obtained by hydrolyzing silk protein, wool, collagen, hair and other natural proteins with an acid or alkaline aqueous solution contains multiple amino acids that make up the protein, use them as an amino acid-containing aqueous solution. You can also That is, when an amino acid containing aqueous solution obtained contains the plurality of amino acids, or a protein hydrolyzate is contacted with the adsorbent, the type of adsorbent selectively adsorbs an acidic amino acid or a basic amino acid , Can be collected. The amino acid-containing aqueous solution is preferably used by adding an acid or an alkali to a predetermined pH range if desired.

To separate and recover a specific amino acid,
For example, a column packed with the adsorbent, the amino acid-containing aqueous solution (neutral or acidic, for example pH = about 5.5) flowed for a predetermined time, the basic amino acid or acidic amino acid is adsorbed on the adsorbent. Next, pure water is caused to flow to wash away the amino acids adhering to the adsorbent. Desorption of adsorbed amino acids can be performed by rinsing with a small amount of acidic aqueous solution (for example, aqueous solution of hydrochloric acid, acetic acid, organic acid, etc. at a specified concentration) in the case of basic amino acids, and removing specific amino acids adsorbed on the adsorbent. It is performed by detaching from the adsorbent. In the case of acidic amino acids, rinse with an alkaline aqueous solution (for example, an aqueous solution of sodium carbonate, ammonium, etc. of a specified concentration),
This is done by removing the adsorbed amino acid. As a result, an aqueous solution containing a large amount of the specific amino acid is obtained. The longer the column packed with the adsorbent, the more effective the adsorption. In addition, the aqueous solution containing a large amount of the specific amino acid adsorbed on the column, obtained by rinsing with an acid or alkaline aqueous solution, is passed through the column again to improve the separation from other amino acids and finally obtain The purity of the specific amino acid used can be increased. In order to confirm the separated state of amino acids by this operation, the amino acid aqueous solution that has passed through the column is checked by liquid chromatography according to amino acid analysis specifications.

[0036]

The present invention will be described in detail below with reference to examples and reference examples , but the present invention is not limited to these examples. (1) Synthesis of polyamino acid (a) The amino acids used in the reference example are as follows: L
-Valin (hereinafter abbreviated as Val. Wako Pure Chemical Industries, Ltd.)
Manufactured by Yoneyama Pharmaceutical Co., Ltd., L-glycine (hereinafter abbreviated as Gly), L-alanine (hereinafter abbreviated as Ala, manufactured by Yoneyama Chemical Co., Ltd.), L-leucine (hereinafter Leu). Tokyo Kasei Co., Ltd., L-phenylalanine (hereinafter abbreviated as Phe, Tokyo Kasei Co., Ltd.), L-glutamic acid ester (hereinafter abbreviated as Glu. Ajinomoto Co., Inc.).
Manufactured by Ajinomoto Co., Inc.) and L-aspartic acid (hereinafter abbreviated as Asp). The organic solvents used in Reference Examples are as follows: hexane (Wako Pure Chemical Industries, Ltd.), ethyl acetate (Wako Pure Chemical Industries, Ltd.), acetonitrile (Wako Pure Chemical Industries, Ltd.), tetrahydrofuran. (THF) (manufactured by Wako Pure Chemical Industries, Ltd.), butylamine (manufactured by Wako Pure Chemical Industries, Ltd.).

(B) Synthesis of amino acid NCA: The amino acids used are all L-forms. 0.1 mol of amino acid (Gly (7.51g), Ala (8.50g), Val (11.7g), Phe (16.5g)
g)) or its mixture (total amount 0.1 mol) is finely crushed in a mortar and placed in a 500 mL eggplant-shaped flask,
To this was added 200 mL of fully dehydrated THF. While heating this reaction vessel at 40 to 50 ° C, 0.051 mol of trichloromethyl chloroformate (6.00 mL) (manufactured by Hodogaya Chemical Co., Ltd.) was added and stirred to suspend. The amino acid NCA can be similarly synthesized by adding triphosgene 0.037 mol (10.9 g) instead of trichloromethyl chloroformate. After 1 to 4 hours, the cloudy amino acid suspension becomes almost transparent, so
At this point, the reaction was completed. THF was removed by distillation under reduced pressure, and the reaction solution was concentrated to 20 to 30 mL. If desired, it is desirable to proceed with the concentration until almost dryness.
Add about 100 mL of hexane to the concentrate and
After cooling in a freezer at ℃ for 4-5 hours, crystals of amino acid NCA precipitate in the flask. The crystals were taken out by filtration, and a recrystallization operation using ethyl acetate as a solvent and hexane as a precipitant was repeated 3 to 4 times to obtain purified amino acid NCA crystals. Glu, Asp
Reacts with a mixture of benzyl alcohol and hydrochloric acid to block the hydroxyl group of the amino acid side chain,
-Benzyl-L-glutamate (BLG) or β-benzyl-L-aspartate (BLA) was designated as NCA. In the case of tyrosine or serine, the side chain hydroxyl group itself is benzylated.

(C) Polymerization of amino acid NCA: 0.1 mol of amino acid NCA was dissolved in acetonitrile, 0.0005 mol of butylamine was added as an initiator, and the mixture was allowed to stand at 50 ° C. for 1 week for reaction, and then the reaction mixture was filtered, It was washed with ethyl acetate, filtered, and dried to obtain various synthetic polyamino acids. Based on the charged amounts of the monomer and the initiator, the average degree of polymerization is
Estimated to be 200. When the amino acid NCA synthesized from Val, Gly, Ala, or Phe was polymerized in this manner, powdery polyamino acids composed of the respective amino acid residues were obtained. The infrared absorption spectrum (IR) of the poly (glycine), poly (L-alanine), etc. synthesized in this way was measured by the potassium bromide tablet method (measuring instrument: Shimadzu Fourier transform infrared spectrophotometer). 8500). From the obtained absorption spectra, it was found that poly (glycine) has a β structure and poly (L-alanine) has a molecular form based on a mixture of α helix and β structure.

(2) Production of micronized silk fibroin fiber The cocoon thread was boiled for 40 minutes in a 0.6% aqueous sodium carbonate solution at about 80 ° C. to remove the silk sericin covering the surface of the cocoon thread.
It was washed with water and dried at room temperature to prepare silk fibroin fiber. 3.0 g of silk fibroin fiber prepared in this way
10% by weight sodium carboxymethylcellulose (CM
It was immersed in 25 mL of an aqueous solution of abbreviated as C and manufactured by Wako Pure Chemical Industries, Ltd., thoroughly mixed, and then dried and solidified. The resinous solid thus obtained was placed in liquid nitrogen for 10 minutes and frozen. The frozen sample was immediately made into fine powder by a mechanical pulverizer mill (Y308B, Yamamoto Electric Co., Ltd., rotation speed: 21,000 times / minute). The obtained powder was washed with boiling water to remove CMC. Then, it was filtered and dried to obtain micronized silk fibroin fiber. Observation with a scanning electron microscope revealed that the fine fibers had an average fiber length of 160 μm and a fiber width size of 13.5 μm. It should be noted that silk fibroin fiber cut to about 0.3 mm with scissors can also be used as the amino acid adsorbent.

(3) Production of granular silk fibroin having an average particle diameter of 0.5 to 1.0 mm An aqueous solution obtained by dissolving silk fibroin fiber (10 g) in a 7.5 mol / L lithium bromide aqueous solution (100 mL) was dialyzed with cellulose. After dialyzing with a membrane, the resulting aqueous solution was blown dry and concentrated to about 1/5. Lyophilize this at -20 ° C,
A powdery solid was obtained. This was crushed in a mortar, and silk fibroin particles having a particle size of 0.5 to 1.0 mm were obtained by using two stainless steel chemical sieves having one side of eyes of 1.0 mm and 0.5 mm, respectively. Similar particles were obtained by using calcium chloride instead of lithium bromide.

(4) Method of packing the adsorbent in a column A predetermined amount of the amino acid adsorbent in a dry state is placed on a stainless steel column (601-11643 column manufactured by GL SCIEN) (inner diameter).
4.0mm x length 150mm), then use a liquid chromatography pump to add water / methanol (50/50% by volume) to 50kg /
The column was packed while flowing with a pressure of cm ² and supplementing the adsorbent.

(5) Evaluation of amino acid adsorption performance on adsorbent (a) Amino acid adsorption performance on adsorbent The amino acid adsorption performance on the adsorbent was evaluated as follows. Amino acid adsorbent is a stainless steel column (inner diameter 4.0 mm
× length 150 mm), and then the column was loaded with 9 amino acids (Ala, Val, Leu, Phe, Glu, Asp, Lys, Ser, Gl)
The mixed aqueous solution (stock solution) of y) (pH = 5.5, each amino acid concentration: 1.6 × 10 ⁻⁵ mol / L, or 8.0 × 10 ⁻⁶ mol / L) was sent by the liquid feed pump (flow rate 0.2 mL / min). . The aqueous solution flowing out from the column outlet was collected with a fraction collector (Advantec, SF-2120) at regular intervals. The amino acid concentration contained in each collected fraction was determined by a liquid chromatograph for amino acid analysis.

Further, the amino acid adsorbent was placed in an Erlenmeyer flask, the amino acid-containing aqueous solution was added thereto, the mixture was stirred for about 10 minutes, and after leaving for 1 hour, a part of the amino acid-containing aqueous solution was sampled for amino acid analysis. The amount of amino acid adsorbed on the adsorbent was determined. In addition, 13 kinds of amino acids (Asp, Gl
n, Asn, Glu, Val, Met, Ile, Tyr, Phe, His, Lys, Tr
Aqueous solution containing p, Arg) (pH = 5.5, amino acid concentration: 8.0)
The amount of amino acid was also determined in the same manner as above when ^{x10 -6} mol / L) was used.

(B) Adsorption mol% of amino acid to the adsorbent The column packed with the amino acid adsorbent was charged with amino acid concentration, Cmol
When 50 mL of an amino acid-containing aqueous solution of / mL is flowed, 50 Cmol has passed. The aqueous solution flowing out from the outlet was sampled at regular intervals in the column, and the concentration of contained amino acids was quantified with an amino acid analyzer. From the product of this concentration and the volume of the aqueous solution that had flowed up to that point, the outflow amount (mol number) of the amino acid was obtained. Since this is the sum of the amounts obtained for each fraction, it is an approximate amount (integral value). The value obtained by subtracting the number of moles of outflowing amino acid from the number of moles of amino acid contained in the stock solution is the number of moles of amino acid adsorbed on the column packed with the adsorbent. The percentage of the number of moles of the adsorbed amino acid to the number of moles of the amino acid contained in the stock solution was defined as the adsorption mole%. Also in the case of batch type adsorption using an Erlenmeyer flask, the same determination was performed.

(C) Two amino acid analyzer liquid feed pumps (LC-10AS manufactured by Shimadzu Corporation), column oven (CTO-10A manufactured by Shimadzu Corporation), precolumn for amino acid analysis (Shim-pack ISC-30 manufactured by Shimadzu Corporation (inner diameter) 4.0mm x length 5
0mm), amino acid analysis column (Shimadzu Shim-pack)
A system consisting of Amino-Na (inner diameter 6.0 mm × length 100 mm) and fluorescence detector (RF-10AXL manufactured by Shimadzu Corporation) was used for the standard gradient analysis for amino acid analysis. Buffer A (pH = 3.2: sodium citrate, perchloric acid) and buffer B (pH = 10.0: sodium citrate, boric acid, sodium hydroxide), which are used as usual eluents for amino acid analysis, and as a washing solution A 0.2M-NaOH aqueous solution was used, and a reaction solution containing the following solution A and solution B was used as the reaction solution.
Solution A = sodium hypochlorite aqueous solution (commercial product 10% effective chlorine solution 0.3 mL / L ratio) and solution B = OPA reagent (orthophenylphenol (OPA) 0.08%: OPA 400 mg, ethanol 7)
ml, 2-mercaptoethanol 1 mL, 10% Brij-35 solution 2 m
L) and were made up to 500 ml with borate-carbonate buffer. Brij-35
Is polyoxyethylene lauryl ether 10g water 100mL
I made it by adding. The boric acid-carbonate buffer solution was adjusted to 3 L by adding water to 122.1 g of sodium carbonate, 40.7 g of boric acid, and 56.4 g of potassium sulfate.

(Example 1) Adsorption of amino acid to micronized silk fibroin fiber (column packing method) 0.123 g of micronized silk fibroin fiber obtained according to the production method described in (2) above was added to a stainless steel column (inner diameter: 4 mm).
× length 150 mm), and then add an amino acid-containing aqueous solution to 0.2
60.5 mL was flown at a flow rate of mL / min. The aqueous solution (fraction) flowing out from the column was taken at regular intervals, and the concentration of each amino acid contained therein was quantified by liquid chromatography. Figure 1 shows the relationship between the obtained amino acid concentration (mol / L) and the flow rate (mL) of the amino acid-containing aqueous solution up to that point.
(For Ser, Leu, Phe, Glu, Asp, Lys) and Figure 2 (Gl
y, Ala, Val). Table 1 also shows the concentration of each amino acid in the amino acid-containing aqueous solution (concentration of the undiluted solution), the concentrations of various amino acids contained in the outflowing aqueous solution when the outflow amount from the column was 20.5 mL, and the adsorbent up to the outflow amount of 20.5 ml. The percentage (adsorption mole%) of the number of moles of each amino acid adsorbed on to the number of moles of the amino acid to be contained in 20.5 ml of the stock solution is shown.

(Table 1)

From FIGS. 1 and 2 up to a flow rate of around 40 mL, Lys
However, there is a tendency to preferentially adsorb to the adsorbent. After that, the Lys adsorption force of the adsorbent becomes small, and a part of the adsorbent elutes without adsorbing. Other amino acids tend to adsorb some of Asp and Glu. The amount of adsorbed amino acid at the time of outflow of 20.5 mL in Table 1 was 95% for Lys, which was overwhelmingly large. Asp and Glu adsorbed 27% and 15%, respectively. Other amino acids (Se
r, Phe, Leu, Gly, Ala, Val) has an adsorption mol% of 0 to 5%.
Is in the range. After that, washing with running distilled water and analysis of the washing liquid revealed that all amino acids other than Lys flowed out into the washing liquid. That is, it was found that micronized silk fibroin fiber specifically adsorbs Lys (basic amino acid). After washing with distilled water, flow about 20 mL of dilute hydrochloric acid aqueous solution (concentration 0.001 mol / L),
The adsorbed Lys could be easily eluted and recovered.

(Example 2) Adsorption of amino acids on micronized silk fibroin fiber (batch system) 0.25 g of micronized silk fibroin fiber was added to Asp, Ser, Glu, Gl.
The mixture was placed in a mixed aqueous solution of y, Ala, Val, Leu, Phe, and Lys (all having a concentration of 8.0 × 10 ⁻⁶ mol / L and a temperature of 25 ° C.) and stirred for 10 minutes. After standing still for 30 minutes, a part of the contacting amino acid-containing aqueous solution was collected, and the type and concentration of the contained amino acid were measured. FIG. 3 shows the concentration of each amino acid before contact with the micronized silk fibroin fiber (that is, the stock solution of the amino acid-containing aqueous solution) and the concentration after contact. Only Lys was found to specifically adsorb. In this case, Lysine 69.7
% Was adsorbed on the silk fibroin fiber (hereinafter, abbreviated as adsorbed silk fibroin fiber). Next, the adsorbed silk fibroin fiber was washed with 50 mL of distilled water to wash away the adhering amino acids, and then the diluted hydrochloric acid aqueous solution (concentration 0.00
The adsorbed amino acid was desorbed by washing with 30 ml of 1 mol / L). Amino acid analysis in this washed diluted hydrochloric acid aqueous solution (recovered liquid) was performed to determine the concentration of the contained amino acids. This concentration is shown in Figure 4.
Shown in. The recovered solution contained almost only Lys. Other amino acids were also contained in trace amounts, but this was because the adsorbed silk fibroin fiber was not sufficiently washed with distilled water, and after thorough washing, only Lys could be recovered. In this way, it became possible to separate and recover Lys by using micronized silk fibroin fiber as an adsorbent.

(Example 3) Adsorption of Amino Acids on Silk Fibroin-Coated Silica Gel Particles The surface of silica gel particles (diameter 5 μm: TSK gel manufactured by Tosoh Corp., Silica-150) was coated with a silk fibroin film as follows. Amino acids were adsorbed onto the silica gel particles thus prepared. 5g of silk fibroin fiber from silkworm at 55 ℃
Was added to 50 ml of 7.5 mol / L lithium bromide aqueous solution and treated for 20 minutes to dissolve the fiber. This aqueous solution was put into a dialysis membrane made of cellulose and replaced with pure water at 10 ° C for 24 hours to obtain a concentration of 1.3.
% Silk fibroin aqueous solution was prepared.

30 mL of the prepared 1.3% silk fibroin aqueous solution
1 g of silica gel particles was put in the flask and stirred at room temperature for 30 minutes, and then the silk fibroin aqueous solution was removed by decantation. Next, the silica gel particles coated with the silk fibroin film were taken out with a spatula and spread on the polyethylene film so as to be as flat as possible. After air-drying for 5-6 hours, the silica gel particles were immersed in a 50% by weight aqueous methanol solution at room temperature for 30 minutes to make the coated silk fibroin film on the surface of the silica gel particles insoluble in water to prepare silk fibroin-coated silica gel particles. .

Whether or not silk fibroin was actually attached to the surface of the thus-prepared silica fibroin-coated silica gel particles was confirmed by infrared absorption spectrum measurement. The infrared absorption spectrum 1650cm ^-1, 1525cm ^-1
Shows absorption due to silk fibroin (amide (I) and amide (II) absorption bands, respectively), and it was revealed that silk fibroin was present in a thin film form on the surface of the silica gel particles. Observation with a scanning electron microscope also confirmed the presence of deposits based on silk fibroin on the surface of the silica gel particles. Column of 1.84 g of silk fibroin-coated silica gel particles (inner diameter 4 mm x length 150 m
m) and filled with an amino acid-containing aqueous solution (concentration = 8.00 × 10 ⁻⁶ mol / L) at a flow rate of 0.2 mL / min in the same manner as in Example 1. The amino acid-containing aqueous solution was allowed to flow to 60.5 ml, and the amino acids contained in each fraction in the middle were analyzed. As a result, it was found that almost all the amounts of Asp and Glu were adsorbed. Table 2 shows the percentage (adsorption mol%) of the amount of each amino acid adsorbed on the silk fibroin-coated silica gel particles to the amount of amino acid in the stock solution when 20.5 ml of the amino acid-containing aqueous solution was passed.

(Table 2)

As is clear from Table 2, Asp and Glu are 100
% Adsorbed. Lys adsorbed 24%. Almost no other amino acids were adsorbed. Then
The silk fibroin-coated silica gel particles thus adsorbed with amino acids were rinsed several times with 100 ml of distilled water. Asp,
Glu could be recovered by desorbing about 5 ml of 0.1 mol / L sodium carbonate (or other alkali) aqueous solution.

(Example 4) Adsorption of amino acid on silk fibroin powder (batch system) Silk fibroin fine powder was produced by the following method. 7.8 g of silk fibroin fiber derived from silkworm was placed in 130 mL of a 4 mol / L calcium chloride aqueous solution (prepared at a ratio of 56 g of calcium chloride, 72 g of water and 46 g of ethanol) and heated to about 70 ° C to dissolve it. The insoluble part in this aqueous solution was removed by centrifugation, and the resulting aqueous solution was put into a cellulose dialysis membrane and kept at about 10 ° C for 2
It was dialyzed against pure water for a day. Air dry the solution in the dialysis membrane,
The volume was concentrated to about 1/5. Next, hydrochloric acid was added to this concentrated solution to adjust the pH to 3.5, and gelled. This gel was frozen overnight to obtain silk fibroin powder, which was immersed in 100 mL of methanol for 1 hour and then dried. This was crushed in a mortar while controlling the force, and then a chemical powder was used to prepare a granular powder having a particle size of about 0.2 to 0.5 mm, which was used as an amino acid adsorbent. 0.5 g of silk fibroin powder thus prepared was added to 10 mL of an amino acid-containing aqueous solution (containing 8.00 × 10 ⁻⁶ mol / L of various amino acids), and stirred in a beaker for 1 hour to adsorb the amino acid. . Next, a part of this amino acid-containing aqueous solution was sampled, and the amino acid concentration contained was determined by an amino acid analyzer. Table 3 shows the concentrations of various amino acids before and after mixing with silk fibroin powder (that is, the amino acid concentration before adsorption and the amino acid concentration after adsorption), and the adsorption mol% of each adsorbed amino acid.

(Table 3)

As is clear from Table 3, only the basic amino acid Lys was selectively adsorbed on the silk fibroin powder. The adsorbed Lys was rinsed 5 times with 5 mL of pure water using the adsorbed fibroin powder through a glass filter.
After washing the amino acids with low adsorptive power, rinsing with 5 mL of hydrochloric acid aqueous solution of pH = 3.0, all Lys is desorbed in the hydrochloric acid aqueous solution,
I was able to recover it.

Reference Example 1 Amino Acid Adsorption to Wool Keratin Powder Wool keratin powder was prepared as follows from an aqueous keratin solution in which wool fibers were dissolved. Pigments and fats contained in Merino wool (64'S) were converted to benzene / ethanol (50/50
6% in Soxhlet extractor with (vol%) mixed solvent
It was removed by time treatment. Prepare a three-necked flask, and connect a rubber tube from a dry nitrogen cylinder to the one port through a three-way cock to adjust the pH of the reaction system.
The electrode is always inserted in another mouth, and the remaining mouth is used for administration of the required drug. Add 8.18 g of merino wool fiber shredded to a fiber length of about 1 cm in a three-necked flask,
To this, 450 mL of 8 mol / L urea solution was added. Purge with nitrogen gas and aspirator inside the three-necked flask for 15 minutes 45
Decrease the pressure to about mmHg and then rapidly return to atmospheric pressure.
Repeated ~ 4 times. In this way, the air contained between the wool fibers in the three-necked flask was completely removed, and the reaction between the aqueous urea solution and the keratin molecule was efficiently performed. After the nitrogen replacement was completed, 4.8 mL of mercaptoethanol was added as a reducing agent into the three-necked flask, and 8mo
It was left to stand in a 1 / L urea aqueous solution for 2 to 3 hours. Then, about 100 mL
5N-KOH aqueous solution was added in small amounts, and the pH of the mixed aqueous solution in the three-necked flask was adjusted to 10.5. The wool fibers were allowed to completely dissolve at room temperature for 3 hours. Thus, the keratin aqueous solution in which the fibrous wool fibers were dissolved was dialyzed with pure water for 2 days using a cellulose dialysis membrane. After dialysis, it was concentrated by blowing air drying, and if necessary, a necessary amount of pure water was added to prepare a keratin aqueous solution having a predetermined concentration.

To 450 mL of the 0.01% keratin aqueous solution thus prepared, 9.5 g of iodoacetic acid was added at room temperature to carry out the S-carboxymethylation reaction of keratin for 1 hour. 5N
An S-carboxymethyl keratin aqueous solution was obtained by adjusting the pH of the keratin aqueous solution to 8.5 with a -KOH aqueous solution. This aqueous solution was dialyzed against pure water for 1 day using a dialysis membrane made of cellulose. The S-carboxymethyl keratin aqueous solution thus produced was freeze-dried to produce a powdery keratin sample. Powderize this using a mortar,
Furthermore, a chemical sieve (stainless steel sieve with a hole diameter of 1 mm and 0.5 mm) is used to remove coarse and fine particles, and a diameter of 0.5
A granular powder of ˜1.0 mm was obtained. A 2% glutaraldehyde aqueous solution was allowed to act on this powder at 25 ° C. for 10 minutes, and finally, it was thoroughly washed with water. This was centrifuged under the same conditions as in Example 4 and dried to prepare a wool keratin powder. The obtained wool keratin powder was packed in a column in the same manner as in Example 1 and used as an amino acid adsorbent. When a predetermined amount of an amino acid-containing aqueous solution was flowed and the amount of amino acids contained in the entire aqueous solution coming out of the column was measured, it was found that Asp and Glu were preferentially adsorbed.

Reference Example 2 Adsorption of amino acids to cut wool keratin fibers 0.246 g of wool keratin fibers finely cut and cut were packed in a column (internal diameter 4 mm × length 150 mm) and amino acid-containing in the same manner as in Example 2. The aqueous solution was flown at a flow rate of 0.2 mL / min. The concentrations of various amino acids contained in the outflowing aqueous solution when the total amount of the amino acid-containing aqueous solution flowing out from the column was made to reach 20.5 mL were quantified by the same method as in Example 1. The amounts of amino acids contained in the aqueous solutions of the stock solutions containing various amino acids and the effluent solutions, and the adsorption mol% of each amino acid adsorbed on the amino acid adsorbent were determined. It was found that Asp and Glu were preferentially adsorbed to the adsorbent on the micronized wool keratin fibers.

( Reference Example 3 ) Adsorption of amino acid to poly (glycine) powder As in Example 1, 0.492 g of poly (glycine) powder was packed in a column (internal diameter 4 mm x length 150 mm) and 0.2 mL of an amino acid-containing aqueous solution was packed. 37 mL was applied to the column at a flow rate of / minute to analyze the amino acids contained in each fraction in the middle. Table 4 shows the percentage (adsorption mol%) of the concentration of each amino acid adsorbed on the poly (glycine) powder in the stock solution when 20.5 mL of the amino acid-containing aqueous solution was passed.

(Table 4)

As is clear from Table 4, Asp and Glu are almost
It was found to adsorb 100%. 0 for other amino acids
The adsorption mol% was about 4.8%. The poly (glycine) powder thus adsorbed with the amino acid was added to distilled water 100
Rinse several times with mL. Asp and Glu could be recovered by flowing about 5 ml of a 0.1 mol / L sodium carbonate (or other alkali) aqueous solution.

( Reference Example 4 ) Adsorption of amino acid to poly (L-alanine) powder As in Example 1, 0.396 g of poly (L-alanine) powder was packed in a column (internal diameter 4 mm x length 150 mm) to contain amino acid. 70 mL of the aqueous solution was passed through the column at a flow rate of 0.2 mL / min, and the amino acids contained in each fraction in the middle were analyzed. In Table 5,
At the time when 20.5 mL of the amino acid-containing aqueous solution was flown, poly (L
-Alanine) shows the percentage (adsorption mol%) of the concentration of each amino acid adsorbed on the powder in the stock solution.

(Table 5)

As is clear from Table 5, Asp is 65%, Glu
Adsorbed 59%. Almost no other amino acids were adsorbed. In this way, poly (L
The alanine) powder was rinsed several times with 100 ml of distilled water. Asp,
Glu could be recovered by running about 5 ml of a 0.1 mol / L sodium carbonate (or other alkali) aqueous solution.

( Reference Example 5 ) Adsorption of amino acid to poly (L-valine) powder As in Example 1, 0.40 g of poly (L-valine) powder was packed in a column (internal diameter 4 mm x length 150 mm) and an amino acid-containing aqueous solution was prepared. 70 mL was applied to the column at a flow rate of 0.2 mL / min, and the amino acids contained in each fraction in the middle were analyzed. Table 6 shows the percentage of each amino acid adsorbed on the poly (L-valine) powder with respect to the concentration in the stock solution (adsorption mol%) when 20.5 ml of the amino acid-containing aqueous solution was passed.

(Table 6)

As is clear from Table 6, poly (L-valine) was an adsorbent similar to those in Example 3 and Reference Examples 1 to 4 and showed specific adsorption of Asp and Glu. That is,
Asp adsorbed 44% and Glu adsorbed 45%. Almost no other amino acids were adsorbed. The poly (L-valine) powder thus adsorbing the amino acid was rinsed several times with 100 mL of distilled water. For Asp and Glu, by flowing about 5 mL of 0.1 mol / L sodium carbonate (or other alkali) aqueous solution,
I was able to recover it.

Reference Example 6 Adsorption of Amino Acid on Poly (L-phenylalanine) Powder Poly (L-phenylalanine) powder was prepared in the same manner as in Example 1.
0.40 g was packed in a column (internal diameter 4 mm × length 150 mm), and the amino acid-containing aqueous solution was flown through the column. The concentrations of various amino acids contained in the outflowing aqueous solution when the total amount of the amino acid-containing aqueous solution flowing out from the column was 5.5 mL were quantified by the same method as in Example 1. Table 7 shows the amounts of amino acids contained in the aqueous solutions of the stock solutions containing various amino acids and the outflowing solution, and the adsorbed mol% of each amino acid adsorbed on the amino acid adsorbent.

(Table 7)

As is clear from Table 7, Asp and Glu are more easily adsorbed than other amino acids. Adsorbed As
p and Glu are weakly alkaline aqueous solutions (sodium carbonate aqueous solution)
It was possible to recover by pouring.

( Example 5 ) Adsorption of amino acids on micronized silk fibroin fiber 0.4098 g of micronized silk fibroin fiber was placed in a glass tube (inner diameter: 6 mm).
× 20 cm in length), an amino acid-containing aqueous solution containing 13 kinds of amino acids was allowed to flow naturally from the top of the glass tube without applying pressure. The concentrations of various amino acids contained in the effluent were quantified in the same manner as in Example 1 when the total amount of the amino acid-containing aqueous solution flowing out of the glass tube was 8.0 mL. Table 8 shows the amount of amino acids contained in the stock solution containing various amino acids and the effluent solution, and the adsorption mol% of each amino acid adsorbed on the amino acid adsorbent.

(Table 8)

As is clear from Table 8, 100% of the basic amino acids Lys and Arg are adsorbed, and His having a stronger basicity than these.
Adsorbed almost 38%. After adsorbing 50 mL of distilled water on the glass tube and washing it, 5 mL of dilute hydrochloric acid aqueous solution was flowed to collect the adsorbed Lys, Arg, and His. A mixture of only these basic amino acids could be separated into individual amino acids using a cation exchange resin after adding a small amount of weak acid to form His as a salt, and then recovered.

Reference Example 7 Adsorption of Amino Acid to Copolymer Each amino acid NCA was dissolved in acetonitrile so that Gly NCA and Ala NCA were contained in a molar ratio of 1: 2. Using butylamine as the polymerization initiator, the amino acid NCA of (3) above
According to the method described in the section "Polymerization method", the mixture was allowed to stand at 30 ° C for 1 week to proceed with the polymerization to synthesize a copolypeptide consisting of residues of Gly and Ala. The obtained copolypeptide was used as an amino acid adsorbent, and packed in a column in the same manner as in Reference Examples 3 to 6 above, and an amino acid-containing aqueous solution was run. The copolypeptide specifically adsorbed Asp and Glu. Adsorbed
Asp and Glu are alkaline aqueous solutions (sodium carbonate aqueous solution)
It was possible to recover by pouring.

Reference Example 8 0.2 g of the synthetic polyamino acid (poly (L-alanine) of Reference Example 4 above) was dissolved in 10 mL of trifluoroacetic acid and concentrated under reduced pressure distillation to about 1/3 volume, followed by chloroform. Was added to give a solution. The insoluble matter was filtered off with a glass filter, and silica gel particles were put into this and stirred. These particles were dispersed in methanol or ethanol and stirred. The synthetic polyamino acid was attached to the surface of the particles. The particles were dried under reduced pressure to prepare silica gel particles whose surfaces were coated with poly (L-alanine). The silica gel particles were packed in a column in the same manner as in Reference Examples 3 to 6 above, and an amino acid mixed aqueous solution containing various amino acids was run. The silica gel particles specifically adsorbed Asp and Glu. The adsorbed Asp and Glu could be recovered by flowing an alkaline aqueous solution (sodium carbonate aqueous solution).

[0078]

Effects of the Invention According to the present invention, the silk protein and the
By using the carrier fine particles whose surface is coated with silk protein as an amino acid adsorbent, a specific amino acid can be adsorbed and recovered. With this adsorbent,
Basic amino acids such as amino acid, arginine and histidine
Can selectively adsorb, aspartic acid, glutamine
Can also selectively adsorb acidic amino acids such as acids
It The form of the amino acid adsorbent of the present invention is powder,
Any of beads, fibers, films and the like can be used. Basic amino acids can be efficiently adsorbed by filling a column with finely cut silk fibroin fibers or powdered silk fibroin sample and flowing an amino acid-containing aqueous solution. Then, by washing with a dilute aqueous hydrochloric acid solution, the adsorbed basic amino acid can be selectively desorbed and recovered. A specific amino acid is effectively adsorbed to the adsorbent and can be recovered by simply placing the adsorbent in an appropriate container instead of the column, adding the amino acid-containing aqueous solution into the adsorbent, and stirring.

[0079] In the present invention, flow glycine, amino acid residues and serine alanine, for use as an adsorbent a large amount inclusive silk protein long amino acid residue side chain, such as tyrosine, an amino acid-containing aqueous solution such as column When,
When using silk protein, together with basic amino acids ,
Acidic amino acids can also be specifically adsorbed and further recovered. The silk protein according to the present invention may be in the above-mentioned form, or may be water-insoluble obtained by coating the surface of a packing material for packing a known column with the silk protein . In the present invention, since the material itself such as silk protein has a function of adsorbing a specific amino acid, it is not necessary to strictly align the particle sizes as in a general column packing material, and the packing method for a column is also strict. It has the great advantage that it is not required. Further, the adsorbent of the present invention has an advantage that it can be manufactured at low cost.

When a column filled with the adsorbent of the present invention is flowed with an aqueous solution containing various amino acids obtained by hydrolyzing natural protein materials such as wool, collagen, hair and silk protein, basic amino acids and acidic amino acids are obtained. Amino acids can be efficiently adsorbed and further recovered. Therefore, the adsorbent of the present invention can be used as a separation material for separating a specific amino acid from a substance mixture system in the chemical industry, the food industry process, etc., and also in the latest science such as material science and biotechnology. Widely available.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the flow rate (mL) of an amino acid-containing aqueous solution and the amino acid concentration (mol / L) in the effluent aqueous solution.

FIG. 2 is a graph showing the relationship between the flow rate (mL) of an amino acid-containing aqueous solution and the amino acid concentration (mol / L) in the effluent aqueous solution.

FIG. 3 is a graph showing the concentration of each amino acid before contact with the micronized silk fibroin fiber and the concentration of amino acid after contact.

FIG. 4 is a graph showing the concentration of each amino acid in the recovered liquid.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI C07C 227/40 C07C 227/40 229/06 229/06 229/24 229/24 229/26 229/26 (58) Fields surveyed (Int. Cl. ⁷ , DB name) B01J 20/00-20/34

Claims (6)

(57) [Claims] 1. A plurality of amino acids comprising a silk protein or carrier fine particles whose surface is coated with the silk protein.
Existing in an aqueous solution containing
Basic amino acids consisting of nin and histidine, and mainly
Then, an acidic acid consisting of aspartic acid and glutamic acid
An amino acid adsorbent for selectively adsorbing mino acids . 2. The amino acid adsorbent according to claim 1, wherein the silk protein is in a powder form, a bead form, a film form, a fibrous form, or a liquid form. 3. A powder or micronized fiber prepared by coating silk protein with a water-soluble resin, solidifying, and then drying or freeze-solidifying, and crushing the silk protein in a dried or freeze-solidified state. The amino acid adsorbent according to claim 1, wherein 4. The silk protein is silk fibroin fiber.
Silk fibers prepared by immersing fiber in a neutral salt solution and heating it to dissolve
Silk fibroin aqueous solution dialyzed against pure water
Add acid to the aqueous broin solution to adjust the pH to 3 to 3.5 and gel
After freeze-drying, freeze-dried powdered silk protein,
Or silk proteins in the silk gland
The amino acid adsorbent according to claim 1 . 5. The amino according to any one of claims 1 to 4.
Contact an acid adsorbent with an aqueous solution containing multiple amino acids.
The amino acid-containing aqueous solution containing the
A basic amino acid consisting of gin, arginine and histidine
Acid, mainly aspartic acid and glutamic acid?
Selectively adsorbing the acidic amino acid consisting of
Then, after washing the adsorbent with water, an acidic aqueous solution is used to
As a salt consisting of lysine, arginine and histidine
The basic amino acid is desorbed from the adsorbent and recovered, and
Aspartic acid and
Acid amino acid consisting of bismuth and glutamic acid is removed from the adsorbent.
A specific amino acid cycle characterized by being separated and recovered
Collection method. 6. The pH of the acidic aqueous solution is 1.0 to 4.0,
Also, the pH of the alkaline aqueous solution is 7.5 to 13.0.
The method for recovering a specific amino acid according to claim 5, which is used as a characteristic.